Device for protein crystallisation

ABSTRACT

The invention relates to a container ( 1 ) with a base body ( 2 ), comprising a base plate ( 3 ) and side walls ( 4 ) standing out therefrom in an at least approximately perpendicular arrangement, and with wells ( 5 ) disposed in the base body ( 2 ). The wells ( 5 ) are provided in the form of a recess in the base plate ( 3 ) and the side walls ( 4 ) of the base plate ( 3 ) are disposed in at least the approximately opposite direction from the recesses in order to accommodate a volume.

CROSS REFERENCE TO RELATED APPLICATIONS

Applicants claim priority under 35 U.S.C. §119 of Austrian ApplicationNo. A668/2002 filed on Apr. 30, 2002. Applicants also claim priorityunder 35 U.S.C. §365 of PCT/EP03/04454 filed on Apr. 29, 2003. Theinternational application under PCT article 21(2) was not published inEnglish.

The invention relates to a container with a base body comprising a baseplate and side walls standing up therefrom in an at least approximatelyperpendicular arrangement and with wells disposed in the base body, aunit for dividing a volume of a container into part-regions and acrystallization device, as well as the use of the container, the unitand the crystallization device.

From sequencing the human genome, it is now known that there areapproximately 30,000 human genes. This corresponds to approximately 1%of the human genome. Every gene in the human genome in turn encodes morethan one protein. It is therefore assumed that there are between 200,000and 500,000 proteins, although only a fraction of the proteins isexpressed in a specific cell type. In addition, proteins are alsosubject to post-translational changes. These take place before theproteins unleash their ultimate biological function.

In the meantime, it has been learned that even slight proteinmodifications and changes in the nature of protein interactions andprotein localization can have a dramatic effect on the cell physiology.The conformation of a protein is also an essential factor inunderstanding the role which it plays. The form of a protein is also ofgreat relevance in pharmacological terms because most pharmaceuticalsunleash their effect on the basis of their capacity to interact with aspecific protein molecule.

In order to understand a bio-macro molecule at atomic level, it isnecessary to know what its three-dimensional structure is. The bestmethod of obtaining structural information about the bio-macro moleculeis through X-ray crystallography. This enables the structure of acrystal to be determined by measuring the reflection and bending ofX-rays in the planes of the lattice network. Another method of analyzingprotein structure, nuclear magnetic resonance spectroscopy (NMR), islimited to smaller proteins, at present up to a maximum 200 to 300 kDa,from solutions. In the case of X-ray crystallography, there is no upperlimit to the size of the proteins which can be analyzed, providedappropriate crystals can be obtained.

The morphology of a crystal is regarded as the result of an intrinsicregularity. Organic materials can also form crystals. However, organicmolecules and proteins are found in crystalline form only very rarely innature and conditions therefore have to be found in which proteincrystals can be grown in order to determine their structure. Someproteins crystallise very readily, whilst in the case of others, findingsuitable crystallisation conditions is very time-consuming. To thisdate, there are some proteins which it has not been possible tocrystallise.

Protein crystallisation is often also described as an art because acrystal grower not only requires enormous perseverance and patience, butalso a lucky hand.

Most bio-macro molecules, e.g. proteins, are only active if thepolypeptide chains are present in their native, correctly coded state.They must therefore be crystallised from aqueous solutions underconditions in which they will not be denatured. Crystallization isinitiated by adding an appropriate precipitation agent, which leads toover-saturation and thus causes the protein to precipitate orcrystallise. All crystallisation experiments depend on a number ofparameters, such as pH value, concentration of the precipitation agent,temperature, ion concentration, ligands, etc., for example.Crystallization will also not occur unless the right combination ofspecific parameters prevails. Every protein behaves individually anddifferently and optimum crystallisation conditions vary from one toanother, which means that they are found either by chance or as a resultof an enormous amount of work.

Proteins carry amino acids with charged ion groups on their surface. Itis thus necessary to find conditions under which the protein moleculesslowly associate with one another in sequence. In the case of proteincrystallisation, this can be achieved by slowly changing the quantity ofwater in which the protein is dissolved, e.g. by means of inorganicsalts. The salt binds water molecules when it dissolves because itsurrounds itself with a sheath of water so that this water moleculeextracts the protein. Once the saturation point is exceeded, the proteinstarts to precipitate. Crystallization can also be obtained using thereverse process, whereby the quantity of water is increased, which thenenables a hydrophobic enzyme to be crystallised. In addition to salts,surface-active substances such as the detergent, polyethylene glycol,can be used as a means of acting on the protein hydrate sheath. In orderfor the protein to finally crystallise, the pH setting must be correctand the temperature must be correct. Protein crystals contain numerouswater molecules in the crystal lattice (30% to 70%). If they weretreated in the same way as crystals of low-molecular organic orinorganic substances, they would dry out—after which the entire crystalcould end up being damaged.

A structural analysis will produce an image of the content of theelementary cell in the form of an electron density because the X-ray isbent by the electrons. How accurately and in what detail the electrondensity can be determined depends on the spatial resolution andultimately the quality of the measured crystal. Irregularities andstructural faults in the crystal reduce the resolution.

Methods have been devised for testing as many different crystallisationparameters as possible. In situations where a large number of proteinshas to be screened in order to find a crystal suitable for X-ray crystalanalysis, it is necessary to automate the crystallisation experiment.

The widely available Linbro plate used for protein crystallisationcontains 24 wells, each with a well capacity of approximately 3 ml,which then have to be individually closed with 24 glass covers. Althoughthe first robots designed to automate crystallisation experiments withthis plate are now on the market, they do not meet the requirements forthe high throughput screening necessary for three-dimensional proteinstructure analysis. Another disadvantage of the Linbro plate is that itsformat does not conform to the standardised micro-titre plate format,which makes very cost-intensive adjustments to the robot systemnecessary in order to work with this format.

Crystallization plates with 24 wells have proved particularly effectiveas a means of crystallising proteins using the so-called “hanging drop”and “sitting drop” methods. In view of the fact that, very often, onlysmall quantities of purified protein (a few milligrams) are available,the “hanging drop” method is currently the method of choice for proteincrystallisation. A 5 μl to 15 μl drop of concentrated protein solutionis placed on a microscope slide with appropriate precipitationsubstances. This glass slide is then placed upside down above areservoir containing approximately 500 μl of a concentrated solution ofprecipitation substances without proteins, so that the drop with theprotein solution is positioned directly above the reservoir solution(retained on the slide by adhesion forces). The glass cover forms a sealtogether with silicone oil applied to the well so that the system ishermetically sealed from the outside. As equilibrium is reached betweenthe two solutions, the protein can crystallise.

As yet, the crystallisation mechanism is not fully understood and agreat deal of effort is needed in order to describe the structure ofbio-macro molecules. Micro-plates with 24 wells for crystallisation areknown from the prior art, although the degree of automation availablefor these plates is very low because standardised automation tools aredesigned for micro-plates with 96 wells or a multiple thereof.Automation is therefore only possible using expensive, speciallydesigned pipette and detection systems. Furthermore, only a small number(namely 24) of bio-macro molecules can be analyzed with these plates.These plates are primarily designed for crystallising bio-macromolecules by the “hanging” and “sitting drop” methods and can not beanalyzed by means of the crystallisation under oil method (micro-batchmethod).

Accordingly, the objective of the invention is to propose a way ofincreasing the degree of automation which may be used in analyzingbio-macro molecules. One particular part-objective of the invention isto propose a device for crystallising bio-macro molecules, which enablesa plurality of different parameters to be tested simultaneously.

This objective is achieved by the the container in accordance with theinvention. The fact that the wells are provided in the form of recessesin the base plate has proved to be an advantage because the level of thecovering hydrophobic liquid does not need to be as high. Firstly, thislow level of hydrophobic liquid prevents the liquid seeping along theside walls of the container due to capillary action, thereby avoidingany risk of contamination, for example due to work surfaces that havenot been cleaned, both for the laboratory personnel and for otheranalyses. Secondly, costs are reduced as a result of the smaller amountof hydrophobic liquid needed. A lower level of hydrophobic liquid isalso conducive to the crystallization behavior of the bio-macromolecules because the crystallization process starts more quickly.Another advantage is the fact the volume of both the precipitationsolutions needed for the reaction and the bio-macro molecule to beanalyzed is smaller.

In accordance with an embodiment of the invention, the fact that thewells have the same disposition, identical movements parallel with thesupport surface are advantageously always effected relative to the samereference points of the wells, even for different wells, when thecontainer is manipulated on an automated basis.

Also of advantage is the fact that many different bio-macro moleculescan be analyzed at the same time, in accordance with an embodiment ofthe invention.

In an advantageous embodiment, the standardized layout of the conical orcylindrical wells makes for a container of a design which is veryeconomical on space. Particularly good use is made of all the availablespace by the wells.

Also of advantage is an embodiment whereby only a small quantity has tobe used for the reaction. Using small quantities means that an analysisis particularly cost effective.

Another embodiment is of advantage because the regular layout of thewells makes the container very easy to process. The process of fillingthe wells is facilitated and the sample can be easily observed duringprocessing and harvesting of the crystals.

Another embodiment has proved to be of particular advantage because theformation of bio-macro molecules can be improved by means of a surfacetreatment.

The design of the container of another embodiment has advantages,whereby the wells are curved in an at least approximately convexarrangement, the advantage of which is that liquid drops are more easilyaccommodated in wells of this shape and the drops migrate more readilyto the center of the well. When placing a liquid drop in position, thesurface tension due to the adhesion between the walls of a recess andthe boundary surface of the liquid drop has less of a counteractingeffect, the more closely the shape of the recess conforms to theapproximately spherical curvature of a drop of liquid.

The advantage of another embodiment is that it facilitates automatedprocessing of the container and in particular of the wells for thepurpose of crystallizing bio-macro molecules.

Another embodiment has also proved to be of advantage because it enablesa visual control during the work process and during incubation of thecontainer. Consequently, once the reaction mixture has been prepared, itis easier to intervene in the reaction.

Also of advantage is an embodiment whereby the partially non-transparentdesign prevents stray light which might distort the measurement resultsduring detection.

Also of advantage is an embodiment whereby the drops in the wells areprevented from shifting or sliding, even though their walls are of aminimum height of only 0.1 mm, for example, thereby ensuring that thebio-macro molecule remains in the same position during the reaction andanalysis process.

Another embodiment is of advantage because the materials are resistantto organic solvents such as acetone, benzene, acetonitrile, dioxan,2,2,2 trifluoroethanol, for example. They are also compatible withvarious salts, buffers and polymers which are frequently used forcrystallization purposes. Polypropylene and cyclo-olefin co-polymers(COC) are also less permeable to water vapor and therefore lesssusceptible to the effect of evaporation than containers made frompolystyrene, for example.

Also of advantage is an embodiment defined whereby the use of differentplastics to make the container enables different properties to beobtained. For example, the wells need only be resistant to stress causedby organic solvents, whereas the side walls do not have to satisfy thisrequirement.

In another embodimentls, the container is easy to orient when workingwith it. Furthermore, by marking the container accordingly, an internalcontrol system and orientation system can be incorporated for automatedprocessing.

Also of advantage is another embodiment whereby the selected productionmethod enables the container to be manufactured rapidly andinexpensively.

Another embodiment has proved to be of advantage, whereby thestandardized dimensions of the container enable the jobs which have tobe carried out with the container to be automated. The possibility ofautomating processing means that a large number of samples can beanalyzed simultaneously. Since the layout of the wells conforms to theSBS standard, a very high density of wells is also obtained. Anotheradvantage is the fact that automation enables several wells to be filledat the same time. Yet another advantage of this embodiment is the factthat the same filling and test systems as those used with micro-titreplates can be used for the container proposed by the invention becausethe number of wells is standardized.

An embodiment has proved to be of advantage, whereby the recess can befilled with a liquid, and evaporation of the crystallization reagentsand hydrophobic liquid reduced, which means that the concentrations ofthese reagents can be kept constant during the entire reaction processand analysis process.

An embodiment has proved to be of advantage because it incorporates atleast one retaining element, which means that the unit for dividing avolume of a container into part-regions has to be placed in a predefinedposition, thereby ensuring that the experiment is more readilyreproducible.

The objective of the invention is also independently achieved by theunit in accordance with the invention. The advantage of this approach isthat by placing the unit in the container, fluctuations in the liquidlevel can be kept to a minimum when transporting the container. Anotheradvantage is the fact that fluctuations in the hydrophobic liquidcoating the wells can be compensated. During both manual and automatedprocessing of the container, it is constantly susceptible to slightvibrations, which can shift the position of the bio-macro molecule andcould thus disrupt the crystallization process. Using the unit ensuresthat the positioning of the bio-macro molecule in the recess can be keptconstant during the entire crystallization process, which can lastseveral days and even a few months, even if the container has to bemoved from one place to another several times.

Another advantage is the fact that, because it acts like a breakwater,processing can be efficiently automated at a higher speed. The wells cannevertheless be thoroughly coated with the hydrophobic liquid becausethe webs are disposed at a distance from the support surface and theconcentration of hydrophobic liquid in each recess and in all the wellsremains constant.

This being the case, an embodiment is of advantage, whereby greaterstrength can be imparted to the unit. As a result of the increase instrength, the unit can be manipulated during the experiment.Furthermore, once it has been thoroughly cleaned, it can be used severaltimes for different experiments.

As a result of another embodiment, a flow connection can be establishedbetween the part-regions. As a result of this flow connection,differences in concentration between the part-regions can becompensated, thereby guaranteeing the same reaction conditionsthroughout the entire container.

In another embodiment, conformity is achieved in the layout of the wellsin the container, thereby facilitating automated processing.

This being the case, another embodiment ther has proved to be ofadvantage because the fact that the webs are spaced at a sufficientdistance from the support surface enables the quantity of hydrophobicliquid to be distributed over the entire base plate of the container,thereby establishing a flow connection between the individualpart-regions.

Another embodiment has proved to be advantage, whereby a fluid-tightbarrier for the hydrophobic liquid is obtained when the webs are placedagainst the base plate. Consequently, when the webs are placed againstthe base plate, many different hydrophobic liquids, such as silicone oilor paraffin oil can be tested on a base plate or, for example, manydifferent ratios of the hydrophobic liquid can be tested relative to oneanother, such as silicone oil to paraffin oil in a ratio of 1:1 or 1:2or 2:1, etc. Another advantage is the fact that by creating thepart-regions, a row can be used for diluting different reagents involvedin the reaction, for example the precipitation solution, for testpurposes.

Another embodiment is also of advantage because the fact that the unitis spaced apart from the side walls makes it easier to insert the unitin and remove it from the container. Another advantage is the fact thatbecause spacers can be used, the frame does not have to be exactlyadapted in the container.

Also of advantage is an embodiment whereby the unit is made easier tomanipulate, especially easier to insert in and remove from thecontainer.

The objective of the invention is also independently achieved as aresult of the crystallization device in accordance with the invention.The advantage of this approach is that both reagent consumption andconsumption of the bio-macro molecules can be reduced to a minimum.Another advantage is the fact that experiments with the same startingsubstance can be repeated several times, thereby obviating the need forlengthy processes to produce more of the bio-macro molecule. Also ofadvantage is the fact that the same bio-macro molecule obtained from anisolation process can be used several times, thereby ruling outdifferences in the analysis results due to different isolationconditions.

The objective of the invention is also independently achieved by usingthe container in accordance with the invention. The advantage of thisapproach is that a plurality of proteins can be crystallizedsimultaneously under the same conditions.

The objective of the invention is also independently achieved by usingthe device in accordance with the invention. The advantage of thisapproach is that the unit acts in the manner of a breakwater for thehydrophobic liquid.

The objective of the invention is also independently achieved by usingthe crystallization device in accordance with the invention. Theadvantage of this is that the large number of crystallizationexperiments which can be carried out in a short time prevents anydegradation of the bio-macro molecule. Furthermore, various differentcrystallization conditions for a bio-macro molecule can be testedsimultaneously.

In order to provide a clearer understanding, the invention will beexplained in more detail below with reference to the appended drawings,which provide simplified, schematic diagrams of a container for holdingsamples.

The invention will be explained in more detail with reference toexamples of embodiments illustrated in the appended drawings.

Of these:

FIG. 1 shows a container with 96 wells for holding samples;

FIG. 2 shows a container with 384 wells for holding samples;

FIG. 3 shows a container with 96 wells incorporating a retaining elementfor a unit;

FIG. 4 shows a container with 384 wells incorporating a retainingelement for a unit;

FIG. 5 shows a unit for dividing a volume, with lateral spacers disposedin a 4×4 pattern;

FIG. 6 shows a unit for dividing a volume, with lateral spacers disposedin a 6×4 pattern;

FIG. 7 shows a unit with cylindrical spacers;

FIG. 8 shows a section through a unit with plate-shaped spacers;

FIG. 9 shows a section through a unit for dividing a volume;

FIG. 10 shows a plan view of a crystallisation device;

FIG. 11 shows a side view of a conical well with a semi-spherical base;

FIG. 12 shows a side view of a cylindrical well with a flat base;

FIG. 13 shows a side view of a cylindrical well with a semi-sphericalbase;

FIG. 14 shows a side view of a cylindrical well with a conical base;

FIG. 15 shows a side view of a conical well with a flat base;

FIG. 16 shows a side view of a conical well with a conical base;

FIG. 17 is an operating diagram illustrating an example of how acrystallisation device is used.

Firstly, it should be pointed out that the same parts described in thedifferent embodiments are denoted by the same reference numbers and thesame component names and the disclosures made throughout the descriptioncan be transposed in terms of meaning to same parts bearing the samereference numbers or same component names. Furthermore, the positionschosen for the purposes of the description, such as top, bottom, side,etc,. relate to the drawing specifically being described and can betransposed in terms of meaning to a new position when another positionis being described. Individual features or combinations of features fromthe different embodiments illustrated and described may be construed asindependent inventive solutions or solutions proposed by the inventionin their own right.

Protein crystallisation permits access to the three-dimensionalstructure of any protein. In addition, it is also in a position tobridge the gap between genomic and structural information aboutbio-macro molecules. Not only is the presence of the protein vital toits function, but also its conformation. The structure of the proteinalso provides important information about interactions with othermolecules.

FIGS. 1 and 2 show a plan view of a container 1 with a base body 2,consisting of a base plate 3 and side walls 4. The base plate 3 haswells 5, which are formed by the base 6 and the walls 7. The side walls4 surround the reaction region 8. A container 1, of the type illustratedin FIGS. 1 and 2, is suitable for conducting tests where miniaturizationis of particular importance. For example, it is possible to provide alarge number of wells 5 in a base body 2 of a container 1 specificallydesigned for this purpose, thereby simultaneously obtaining a largenumber of reaction regions 8. FIGS. 1 and 2 illustrate an example of alayout of a plurality of wells 5 in a container 1, that illustrated inFIG. 1 having 96 wells 5 and that illustrated in FIG. 2 having 384 wells5. The base body 2 of the container 1 is designed to the standard sizeof a micro-titre plate and its dimensions conform to the recommendationsof the SBS (Society of Biomolecular Screening; www.SBSonline.org), inwhich case the container 1 has an overall height selected from a rangewith a lower limit of 6 mm, preferably 8 mm, in particular 10.4 mm, andan upper limit of 22 mm, preferably 16 mm, in particular 14.4 mm.

The wells 5 are arranged in a rectangular pattern. Accordingly, therespective adjacent wells 5, which each have their own separate walls 7,are separated from one another in mutually parallel rows. These rows arealigned parallel with the longitudinal extension of the container 1 andthe consecutive rows of wells 5 are disposed at an equal distance fromone another. In an alternative embodiment of a container 1, the rows mayalso be disposed perpendicular to the longitudinal extension.

The wells 5 are arranged in rows. The concept of a row in all casesshould be construed in the geometric sense as being a linear arrangementof identical objects, so that respective identical points of the objectslie on a common straight line. The wells 5 constitute a reaction region8, each well 5 having a separate wall 7. If the wells 5 are disposed ina very dense pattern, adjacent wells 5 may have a common wall 7.

The side walls 4 of the base plate 3 constitute the container supportsurface at their bottom edge. The bases 6 of the wells 5 are disposed ina plane parallel with the support surface. The distance of the bases 6of the wells 5 from the surface 14 of the base plate 3 is shorter thanthe distance of the bottom edge of the side walls 4 of the container 1from the surface 14 of the base plate 3. The bases 6 of the wells 5 aretherefore disposed at a distance selected from a range with an lowerlimit of 0.1 mm, in particular 0.3 mm, preferably 0.5 mm, and an upperlimit of 7 mm, in particular 6 mm, preferably 5 mm. It has also beenfound to be of advantage if the distances are selected from a range withan upper limit of 0.7 mm, in particular 1 mm, preferably 1.5 mm, and anupper limit of 4 mm, in particular 3 mm, preferably 2 mm.

In an alternative embodiment, the base plate 3 may be disposed in aplane parallel with the support surface, on which the base 6 of thewells 5 is also disposed. Accordingly, the base plate 3 and the bases 6of the wells 5 are disposed on the same plane in this embodiment. Thetop edge of the walls 7 of the wells 5 therefore lies in a planeparallel with the support surface but at a greater distance than theplane in which the base 6 of the wells 5 is disposed.

In the reaction region 8, the wells 5 have the capacity to hold a volumeselected from a range with a lower limit of 0.01 μl, preferably 0.5 μl,in particular 0.1 μl, and an upper limit of 50 μl, preferably 10 μl, inparticular 5 μl. The wells 5 have a diameter selected from a range withan upper limit of 10 mm, preferably 7 mm, in particular 5 mm and a lowerlimit of 0.1 mm, preferably 0.3 mm, in particular 0.5 mm. What have beenfound to be of particular advantage are diameters selected from a rangewith an upper limit of 4 mm, preferably 3 mm, in particular 2 mm and alower limit of 0.6 mm, preferably 0.7 mm, in particular 0.9 mm. Theheight of the wells 5 is selected from a range with a lower limit of 0.1mm, preferably 0.2 mm, in particular 0.3 mm and an upper limit of 12 mm,preferably 10 mm, in particular 6 mm. It has been found that heights ofwells selected from a range with a lower limit of 0.5 mm, preferably 0.6mm, in particular 0.8 mm, and an upper limit of 5 mm, in particular 3mm, preferably 2 mm, and more especially of 1 mm, are of particularadvantage.

The container 1 and the wells 5 are designed to hold liquids andsolutions. The container 1 is specifically designed to hold hydrophobicliquids 9 and the wells 5 are specifically designed to hold the drop 10consisting of a bio-macro molecule 11 combined with crystallisationreagents 12, which determine what hydrophobic liquid 9 is used. Thereservoir 13 is filled with the hydrophobic liquid 9 to a level wherebythe hydrophobic liquid 9 also moves into the wells 5 of the container 1during deployment of the container 1 in its position of usage. In otherwords, the entire surface 14 of the base plate 3 of the container 1 istherefore covered with the hydrophobic liquid 9. The wells 5 aredesigned for conducting reactions, which are intended to produce acrystal from bio-macro molecules 11 dissolved in crystallisationreagents 12.

The surface 14 of the base plate 3 may be surface-treated between thewells 5 with a hydrophobic substance, such as grease, in particularsilicone grease, oil, polyethylene glycol, polytetrafluoroethylene, forexample. Alternatively, a hydrophobic mask may also be applied to thesurface 14 of the base plate 3, so that the drop 10 is disposed in thereaction region 8 and the hydrophobic liquid 9 is between the reactionregions 8. As a result of the hydrophobic intermediate spaces, the drop10 remains in the same position of the container 1 during the entireanalysis. The surface 14 of the base plate 3 between the reactionregions 8 may also have a matt structure.

The container 1 is specifically used for crystallising bio-macromolecules 11 under a hydrophobic liquid 10, also known as the“micro-batch method”. Crystallization under oil is a method whereby asmall drop 10 consisting of a bio-macro molecule 11 combined withcrystallisation reagents 12 is pipetted under a layer of oil. The ratioof the volume of the bio-macro molecule 11 to the crystallisationreagents 12 needed for this purpose is usually a ratio by volume of 1:1and the drop 10 comprises a total volume of from 1 μl to 2 μl. However,it is also possible to pipette drops 10 with a volume of up to 5 μl intothe wells 5. The most commonly used hydrophobic liquids 9 are paraffinoil or silicone oil or a mixture of these two oils, usually in a ratioof 1:1. However, other ratios of paraffin oil to silicone oil may alsobe used in order to vary the diffusion rate of the drop (the higher thepercentage of silicone oil, the more rapidly diffusion and evaporationoccur).

The material used to make the base 6 of the wells 5 may optionally betransparent and the base plate 3 may be made from a non-transparent orlight-screening material or with a non-transparent or light-screeningcoating. The light-screening effect prevents any disruptive influencesdue to stray light when visually observing the progress of the reactionin the reaction regions 8. The container 1 may naturally also be madeentirely from a transparent or non-transparent plastic.

FIGS. 2 and 3 show recesses 15, which may be provided both at thelongitudinal side 19 and at the transverse side 20 of the base body 2 oreither on the longitudinal side 19 or on the transverse side 20. Therecess 15 may additionally be split into several smaller part-regions bymeans of dividing walls 16. The purpose of the recesses is to holdliquids, in which case different liquids or different concentrations ofthe same liquid may be placed in each part-region, for example, whichprevents or minimizes evaporation of the crystallisation reagents 12 andhydrophobic liquid 9. The top edge of the boundaries of the recesses 15lies on the same plane as the top edge of the walls 7 of the wells 5.Alternatively, the surface of the recess may also be disposed in aparallel plane, but at a greater distance from the support plane thanthe plane of the top edge of the walls 7 of the wells 5.

FIGS. 3 and 4 show retaining elements 17 disposed on the side walls 4 ofa container 1 proposed by the invention. The retaining elements 17comprise two side parts 18, which may optionally also be joined to thebase plate 3. The side parts 18 are arranged at a distance apart so thata spacer 26 of a unit 22 can be inserted between the two side parts 18of the retaining element 17. The retaining elements 17 may be providedboth on the longitudinal side 19 as well as on the transverse side 20 ofthe side walls 4 of the container 1. Another possibility is to provideretaining elements 17 in the corners 21 of the container 1. A retainingelement 17 of the unit 22 for dividing a volume prevents the unit frominadvertently sliding in the event of the container 1 being moved duringprocessing. Any number of retaining elements 17 may be provided on theside walls 4 of the base body 2. In FIG. 3, for example, eight retainingelements 17 are illustrated, in which case two retaining elements 17 areprovided on each longitudinal side 19 and on each transverse side 20 ofthe base body 2. In FIG. 4, four retaining elements 17 are provided,each being disposed in the corners 21 of the base body 2. The spacers25, which are disposed on the frame 24 of the unit 22, are inserted inthe retaining elements 17 on the side walls 4 or the base plate 3 of thecontainer 1. The unit 22 is therefore flexible and can be removed at anytime and used for other analyses. In an alternative embodiment, the unit22 may also be mounted on the side walls 4 or the base plate 3 of thecontainer 1 without retaining elements 17, for example by means ofadhesive. These layouts of the retaining elements 17 should be construedas examples of how the retaining elements may be laid out. Naturally,any other layout could be selected for the retaining elements 17.

The number of wells 5 in the base body 2 may be selected from a groupcomprising the product of the mathematical formula 3×2^(n), where n is awhole number. Naturally it would also be possible to make containers 1with a different number of wells 5 corresponding to one of the standardsizes of micro-titre plates, such as 24, 48, 96, 384, 1536 etc., forexample.

As seen in plan view, the wells 5 have a circular base surface. Theavailable capacity and the base surface of the base plate 3 is used veryefficiently by the layout of wells 5, as illustrated in FIGS. 1 to 4.Naturally, the base surface of the wells 5 may also be rectangular,square or in the shape of a parallelogram. Another possibility would beto arrange the mutually parallel rows of wells 5 offset from one anotherin the longitudinal direction of the container 1. In another embodiment,it would also be possible to provide wells 5 with a regular hexagonalcross-section and dispose the wells 5 in a honeycomb pattern. The bestdesign of wells 5 for automated processing of the container 1 withstandardised laboratory robots is one with a circular base surface inrows.

The container 1 is preferably made from polystyrene or COC. Naturally,the container 1 may also be made from a different material, bypreference plastics, which are suitable for molding by injection-moldingtechniques, such as polypropylene, acrylo-butadiene styrene, polyamidepolycarbonate, polymethyl methacrylate, polysulphone and/or styreneacrylonitrile, etc. for example. A combination of two or more materialsor different plastics may be used, in which case the COC might be usedfor the wells 5 and polystyrene for the base plate 3. In an alternativeembodiment, the base plate 3 and the wells 5 are made from COC and theside walls 4 from polystyrene.

Markings may be provided in the base plate 3 of the base body 2 or onthe side walls 4 to indicate co-ordinates of the container. The markingsmay be both optically visible or be evident in the form of slightchanges in the surface 14, which can be recognised by an automaticlaboratory manipulation system.

FIGS. 5 and 6 show a plan view of a unit 22 for dividing a volume intopart-regions, the unit 22 being made up of webs 23 disposed atright-angles to one another. Optionally, the layout of the webs 23 maybe in a different geometric pattern, e.g. hexagonal, octagonal, etc. Inorder to improve strength, the webs 23 may be enclosed by a frame 24.The number of webs 23 is variable. For example, the unit 22 may have 16sub-divisions as illustrated in FIG. 5 or 24 sub-divisions asillustrated in FIG. 6. Naturally, the unit 22 proposed by the inventionmay also have any other number of sub-divisions.

Bottom spacers 25 and side spacers 26 may be provided on the frame 24 ofthe unit 22 for placing it in a container 1. The bottom spacers 25 areprovided on the bottom edge 27 of the webs or the frame 24. The sidespacers 26 are disposed respectively on the longitudinal side 28,transverse side 29 and/or in the corners 30 of the frame 24 of the unit22. In FIG. 5 for example, every longitudinal aide 28 and transverseside 29 has two spacers 26 each. FIG. 6 illustrates an alternativelayout of the side spaces 26 at the corners 30 of the frame 24 of theunit 22.

FIG. 7 shows a perspective view of bottom spacers 25 for the base plate3 of a unit 22 proposed by the invention. The spacers 25 may be disposedin the intersecting regions 31 of the webs 23 or on the webs 23 betweenthe intersecting regions 31. The spacers 25, 26 may be cylindrical (FIG.7), plate-shaped (FIG. 8), etc. In the region where they serve asspacers, the height of the webs 23 and the frame 24 is greater by athird to a half than in the regions where they do not serve as spacers.The fact that the unit 22 is retained in the base body 2 by the spacingand retaining system means that the hydrophobic liquid 9 disposed in thecontainer 1 is uniformly distributed across the entire base plate 3. Asa result of using the unit 22, movements of the hydrophobic liquid 9 arenot propagated across the entire base plate 3 with the same intensity ifthe container 1 is vibrated because the unit 22 acts like a breakwater.

FIG. 9 shows a section through a unit 22, in which the webs 23 of theunit 22 are designed to extend continuously as far as the base plate 3.As a result, many separate different reaction chambers 33 can becreated. The hydrophobic liquid 9 placed in each well 5 can therefore beused in a different composition or concentration in each differentreaction chamber 33. For example, this enables a row for diluting thecrystallisation reagents 12 to be provided in order to test thebio-macro molecules 11 and/or the hydrophobic liquid 9. In thisembodiment, the external dimensions of the unit 22 are slightly smallerthan or the same size as the dimensions of the container 1.

The unit 22 may also be placed with the side spacers 26 on the sidewalls 4 of the base body 2. The bottom spacers 25 therefore project onlyinto the reservoir 13 of the container 1. The height of the webs 23 andframe 24 in this embodiment is more than a third to a half higher in theregions where they serve as spacers than in the region where they do notact as spacers. In this embodiment, the external dimensions of the unit22 are at least as big as or bigger than the dimensions of the container1.

FIG. 10 illustrates a plan view of a crystallisation device 32,comprising a container 1 and a unit 22 for sub-dividing a volume in theassembled state.

FIGS. 11 to 16 illustrate various different embodiments of the designused for the wells 5, seen from a side view. The bases 6 of the wells 5may be at least approximately concave in curvature, i.e. the bases 6 orthe base regions of the wells 5 may be designed in the shape of a cone,a truncated cone or cone section or a combination of such bodies bywidening the wells 5. This design of the reaction regions 8 assists thetask of depositing the drop 10. The base 6 of the wells 5 may be madefrom a transparent material, in which case the crystal growth in thereaction region 8 can be observed without the need for manipulation.

FIG. 11 shows a side view of a conical well 5 with a semi-spherical base6.

FIG. 12 shows a side view of a cylindrical well 5 with a flat base 6,FIG. 13 shows a side view of a cylindrical well 5 with a semi-sphericalbase 6 and FIG. 14 shows a side view of a cylindrical well 5 with aconical base 6. FIG. 15 gives a side view of a conical well 5 with aflat base 6 and FIG. 16 shows a side view of a conical well 5 with aconical base 6.

FIG. 17 is an operating diagram illustrating how the crystallisationdevice 32 proposed by the invention is used for the micro-batch method.The reaction region 8 of the wells 5 of the container 1 is filled with adrop 10 comprising bio-macro molecules 11 and crystallisation reagents12, whilst the hydrophobic liquid 9 is disposed in the container 1 andthe wells 5. Diffusion may take place in the wells 5 between the drop 10and the hydrophobic liquid 9. The reaction progress in the wells 5 maybe observed with a microscope. An example of a reaction which can beconducted in a container 1 is one in which a crystal is produced frombio-macro molecules 11 dissolved in crystallisation reagents 12, asexplained earlier in the description. A small quantity of bio-macromolecules 11 combined with crystallisation reagents 12 is pipetted undera layer of a hydrophobic liquid 9. The important factor about thismethod is that the reagents involved in the crystallisation must bepresent in a specific concentration and there must be no significantfluctuations in the concentration of the bio-macro molecule 11 or in thecrystallisation reagents 12 in the drop 10. By varying the hydrophobicliquid 9, which might be paraffin oil and/or silicone oil, or by varyingthe ratio of the substances used to make up the hydrophobic liquid 9, ashift in concentration in the drop 10 can occur due to the diffusion ofwater through the oil. This results in an increase in concentration ofthe bio-macro molecule 11 and crystallisation reagents 12 under thehydrophobic liquid 9. This shift in concentration results in theformation of crystals, in particular single crystals. The crystalsformed in the drop 10 can be observed or detected with the aid of adetection system.

The container 1 may optionally also be provided with a vessel cover.This vessel cover may be made from the same material as the container 1or from a different plastic. In addition to the cover, the container 1may also be provided with a film. This film may be adhered to the sidewalls 4 of the container 1. This film may also be joined to the webs 23of the unit.

For the sake of good order, it should finally be pointed out that inorder to provide a clearer understanding of the structure of thecontainer 1, it and its constituent parts are illustrated to a certainextent out of scale and/or on an enlarged scale and/or on a reducedscale.

The independent solutions to the underlying objective of the inventionmay be found in the description.

Above all, the embodiments of the subject matter illustrated in FIGS. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 constituteindependent solutions proposed by the invention. The associatedobjectives and solutions may be found in the detailed descriptions ofthese drawings.

List of reference numbers 1 Container 2 Base body 3 Base plate 4 Sidewall 5 Well 6 Base 7 Wall 8 Reaction region 9 Hydrophobic liquid 10 Drop11 Bio-macro molecule 12 Crystallisation reagent 13 Reservoir 14 Surface15 Recess 16 Dividing wall 17 Retaining element 18 Side part 19Longitudinal side 20 Transverse side 21 Corner 22 Unit 23 Web 24 Frame25 Spacer (bottom) 26 Spacer (side) 27 Bottom edge 28 Longitudinal sideof the unit 29 Transverse side of the unit 30 Corner of the unit 31Intersecting region 32 Crystallisation device 33 Reaction chamber

1. A container comprising a base body consisting of a base plate andside walls standing up therefrom in an at least almost perpendiculararrangement, wells disposed in the base body which are provided in theform of a recess in the base plate, the side walls being disposed in anat least substantially opposite direction from the recesses in order toaccommodate a volume of the container, and a lattice-type unit withinthe container for sub-dividing the volume into part-regions.
 2. Thecontainer as claimed in claim 1, wherein the wells are arranged in auniform pattern.
 3. The container as claimed in claim 1, comprising 6,12, 24, 48, 96, 384 or 1536 wells.
 4. The container as claimed in claim1, wherein the wells are of a conical or cylindrical design.
 5. Thecontainer as claimed in claim 2, wherein the wells have a capacityselected from a range with a lower limit of 0.01 μl, and an upper limitof 50 μl.
 6. The container as claimed in claim 1, wherein the wells havebases disposed in a plane parallel with a container support surface. 7.The container as claimed in claim 1, wherein the base plate and thewells are disposed in a plane parallel with a container support surface.8. The container as claimed in claim 1, wherein the wells are surfacetreated with aldehyde, silane, epoxy, thiol, polyethylene glycol (PEG),polyoxyethylene-sorbitan-monolaureate, magnetic materials, streptavidinor biotin.
 9. The container as claimed in claim 1, wherein the wells aresquare, rectangular, conical or semi-spherical as seen from a side view.10. The container as claimed in claim 1, wherein the wells are round,quadrangular, hexagonal, octagonal or in the shape of a parallelogram asseen in top view.
 11. The container as claimed in claim 1, wherein thebase plate between the wells is at least partially non-transparent. 12.The container as claimed in claim 1, wherein the surface of the baseplate between the wells is surface-treated with a hydrophobic substanceor has a hydrophobic mask applied to it.
 13. The container as claimed inclaim 1, wherein the base body is made from a material selected from agroup consisting of polypropylene, polystyrene,acrylo-butadiene-styrene, polyamide, polycarbonate, polymethylmethacrylate, polysulphone, cyclo-olefin copolymer, polymethyl penteneand/or styrene-acrylonitrile.
 14. The container as claimed in claim 1,wherein the base body is made from several different materials.
 15. Thecontainer as claimed in claim 1, wherein a co-ordinate marker isprovided for indicating the layout of the wells in the base plate. 16.The container as claimed in claim 1, wherein the base body is preferablymade by an injection-moulding process.
 17. The container as claimed inclaim 1, wherein a recess is provided on a longitudinal side and/or on atransverse of the base body.
 18. The container as claimed in claim 1,comprising at least one retaining element on the side walls of thecontainer for a lattice-type unit.
 19. The container as claimed in claim1, wherein webs of the lattice-type unit are surrounded by a frame. 20.The container as claimed in claim 1, comprising spacers on the bottomface of the lattice-type units to establish a flow connection betweenthe part-regions.
 21. The container as claimed in claim 19, wherein thewebs of the lattice-type units are disposed at right-angles to oneanother.
 22. The container as claimed in claim 19, wherein the height ofthe lattice-type units in intersecting regions of the webs is one thirdto a half higher than it is between the intersecting regions.
 23. Thecontainer as claimed in claim 19, wherein the webs and/or the frame areof identical height.
 24. The container as claimed in claim 19, whereinthe frame has spacers for holding the lattice-type units at a distancefrom the side walls of the container.
 25. The container as claimed inclaim 1, wherein the external dimensions are slightly smaller than thedimensions of the container.
 26. The container as claimed in claim 1,wherein made from a material selected from a group consisting ofpolypropylene, polystyrene, acrylo-butadiene-styrene, polyamide,polycarbonate, polymethyl methacrylate, polysulphone, cylo-olefincopolyrmer, polymethyl pentene and/or styrene-acrylo-nitrile.
 27. Thecontainer as claimed in claim 1, wherein the wells are at leastpartially of a transparent synthetic resin.